1. Field of the Invention
The present invention relates to a thermal cycle apparatus, and more particularly relates to such an apparatus useful for the amplification of nucleic acid sequences, and further relates to a method for thermally cycling samples of material, such as nucleic acid sequences, between lower and higher temperatures in order to amplify the amounts of such materials to facilitate their detection.
2. Background Information
Genetic diseases or disorders, infectious diseases and related disorders may be diagnosed by analyzing targeted nucleic acid sequences in DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). These nucleic acid sequences of interest generally are small segments of the DNA or RNA strands. In performing this analysis for diagnosis of a disease or a genetic disorder, it is appreciated that extremely small amounts of genetic material are all that is available.
For detecting the presence of certain sequences of nucleic acids, a relatively new procedure has been developed which amplifies the targeted sequence. This process is known as polymerase chain reaction (PCR) and was described by Saiki et al., Science 230, 1350-1354 (1985) European Patent Application No. 86 302298.4, published Dec. 10, 1986, and U.S. Pat. No. 4,800,159. In addition to increasing the amounts of DNA or RNA material in order to make nucleic acid sequence more easily detectable, the PCR process enhances the sensitivity of the detection of genetic disorders. For example, by amplifying the targeted nucleic acid sequence, it is possible to study single base changes, analyze the absence of base pairs and determine whether there may be translocations of the nucleic acids within the specific sequence of interest. While PCR is an excellent technique for amplifying nucleic acid sequences, there are other procedures which have been used to provide such amplification.
Briefly, PCR involves a primer-mediated enzymatic amplification of the nucleic acid sequence. For example, to amplify specific DNA segments, the DNA is denatured with a pair of synthetic oligonucleotides which serve as primers for annealing with the single strands of denatured genomic DNA. The synthetic oligonucleotides are then extended with a DNA polymerase and deoxynucleotide triphosphates in order to double the number of nucleic acid sequences between the primers. With repeated cycles of denaturation, primer annealing and extension of the primers, the base pair region between the primers is copied over and over again, resulting in an exponential amplification of the DNA segment of interest. The details of the PCR process are found in the aforementioned publications.
A thermally stable DNA polymerase from thermus aquaticus (Taq) has become available, and when used in PCR, significantly simplifies the reaction, as reported by Weier et al. in DNA, vol. 7, no. 6, 1988. The PCR reaction is significantly simplified because amplification is achieved by repeatedly heating and cooling of samples containing the thermally stable polymerase (Taq), the primers, genetic material to be amplified and the deoxynucleotide triphosphates. According to Weier et al., the Taq polymerase has maximum activity between 60.degree. C. and 85.degree. C., and is not destroyed when heated to 95.degree. C. for several minutes. Since denaturation of the genetic material, DNA, can be accomplished at temperatures in the 91.degree.-93.degree. C. range, the Taq polymerase is not destroyed at these temperatures.
Primer annealing is achieved by cooling the sample of materials, so that there is a rapid change of temperature, from hot to cold, of the genetic materials to be amplified. Repeatedly heating and cooling of samples of genetic material, along with the other ingredients for PCR, require proper equipment, such as a thermal cycle apparatus.
It is often desirable when amplifying DNA segments of interest, in conjunction with selected enzymes such as DNA polymerase, to run through perhaps 15-20 rapid heating/cooling cycles. Present performance is limited by the speed at which available equipment may cycle between the temperature extremes. For example, there is a PCR apparatus marketed by Perkin Elmer Cetus, known as a DNA thermal cycler. In this and other similar cycling systems, small tubes or microtiter trays are loaded onto a metal heating/cooling block which is designed to provide equalized temperatures. Heating is achieved by electric heaters embedded in the heat block; cooling is done either by circulating cool water or by a thermal electric cooler. Heat transfer rates of the existing cycling equipment are limited to thermal changes of less than 1.degree. C./sec.
Such limited speed is attributed to at least two factors. First, the energy delivered to the system must heat or cool the large mass of the heat block, heating rods, and cooling water, before the metal block transfers heat to the sample undergoing amplification. Second, thermal contact between the sample and the heat block usually is poor. Heat must pass through a relatively thick-walled plastic tube, which holds the material to be amplified, which in turn is in relatively loose contact with the heat block.
As a result of the designs of the existing thermal cycling systems, the efficiency of performance and operation is quite low. The combined thermal mass of the heat block, the heater rods and the cooling water exceeds the thermal mass of the sample liquid to be cooled generally by a factor of thirty or more. This means that less than 3% of the energy is used to heat or cool the sample, while the rest of the energy is wasted. Since all of the heat energy delivered to the system must be removed by the cooling water, the cooling apparatus grows way out of proportion to the relatively small samples of material undergoing PCR.
Another apparatus which suffers from the same deficiencies as pointed out above, is described by N. S. Fouikes et al., in Nucleic Acids Research, vol. 16, no. 12, 1988. Other apparatuses for DNA amplification are described by Torgersen et al., in Analytical Biochemistry, 176, 33-35 (1988) and by McGraw et al., in DNA and Protein Engineering Techniques, vol. 1 no. 5, 65-67 (1988).
Rather than use an automated thermal cycle apparatus for PCR, it is known that racks of tubes containing the sample materials have been moved from a cold bath to a hot bath in repeating cycles. This type of arrangement is cumbersome, requires substantially more user attention and has many of the same inefficiencies as the previously described existing equipment.
Improvements are thus required in a thermal cycle apparatus for rapid heating/cooling cycles useful for the amplification of nucleic acid sequences, such as employed in the PCR process. The present invention is directed to such an improved rapid thermal cycle apparatus, the device use therein and methods of use.